To define the mechanism of interferon induction by doublestranded (ds)RNA by exploiting our discovery of the most efficient inducer of interferon known (Nature 266:815, 1977) and a cell system hyperresponsive to both the induction and action of interferon. We propose to test several hypotheses on the mechanism of interferon induction by viruses and dsRNA, and the molecular and cellular events which regulate interferon production and the hyporesponsive state. More specifically we will (i) test further our hypothesis that double-stranded (ds)RNA is the interferon inducer moiety of viruses and that the threshold for induction is one molecule of dsRNA per cell, (ii) determine the minimal length of dsRNA required for induction of interferon, (iii) develop a mathematical model which describes the two different types of dose(multipicity)-response (interferon-yield) curves we have characterized quantitatively, (iv) define the early critical "processing" steps in the cell required to deliver a single molecule of dsRNA as an inducer entity, (v) test our hypothesis that nucleocapsid protein loss precedes dsRNA formation by (+)RNA DI particles, and (vi) test a model we have developed to account for the regulation of interferon production. *** Genetic Cloning of Defective-Interfering (DI) Particles: To isolate and maintain genetically pure clones of both 5'-and 3'-end derived defective-interfering (DI) particles by three new procedures, namely, clonal isolation from (i) individual cells manifesting all-or-none homotypic interference, (ii) single cells killed by DI particle action, and (iii) complementation-dependent plaques. Clones of DI particles will be used immediately in studies on the mechanism of interferon induction and regulation, and for long-term use in determining their role in (i) persistent infection and disease, (ii) the function of host-cell factors in their generation, and (iii) the basis of homotypic and heterotypic interference. ***Cell Killing, Cell Sparing and Persistent Infection: To use singlecell plating procedures and determine which virus genes must be expressed to kill a cell. We will (i) define the role of host-cell factors in cell killing by viruses, (ii) test whether the interferon system controls the expression of some host range mutants, and (iii) test further our hypothesis for the role of interferon in the regulation of virus-cell persistence.